Assay system

ABSTRACT

An assay apparatus comprising: i) an assay cartridge ( 52, 53 ) comprising at least one well ( 57 - 62 ) and a pipette ( 50 ) positionable in at least one said well; ii) a holder arranged to receive said cartridge; iii) drive means operable to position said pipette in selected wells of said cartridge; iv) a gas pressure applicator couplable to said pipette whereby to cause liquid flow through said membrane; and v) a radiation detector operable to detect radiation from a well of said cartridge or from said pipette.

This invention relates to improvements in and relating to assay systems,especially diagnostic assay systems, in particular systems usable at thepoint-of-care, e.g. at the physician's place of work or at the patient'sbedside.

Many diagnostic assays are currently available, e.g. assays forpregnancy, blood sugar, homocysteine, carbohydrate-deficienttransferrin, blood-clotting, blood cholesterol, etc. Some such assaysare performable by the patient, and some by the patient's physician, butmany, especially those which provide a quantitative result, mustcurrently be performed in a laboratory remote from both patient andphysician and so result in significant delays between sampling andtesting and generally require the patient to make a further visit to thephysician to learn the assay's results. This is not only inconvenient tothe patient but also increases the costs to the patient or theorganization paying for the patient's health care.

There is thus an ongoing need for assay systems, especially onesproviding quantitative results, operable by the physician or thephysician's colleagues at the point of patient care.

Quantitative assay systems often require highly accurate volumemeasuring devices, several reagents, and assay-specific result-readingdetectors, and it is impractical to provide, dedicated assay apparatusfor a wide range of different assay systems at the point of care, bothfor reasons of space and expense.

We have therefore developed an assay apparatus which, in preferredembodiments, is capable of use at the point of care, is capable ofperforming a range of different assays, is capable of yieldingquantitative assay results, and is relatively inexpensive.

Viewed from one aspect the invention provides an assay apparatus,preferably a diagnostic assay apparatus, comprising:

i) an assay cartridge comprising at least two wells and a pipettepositionable in at least two of said wells, said pipette having aproximal end and a distal end, said distal end being closed by a liquidpermeable membrane;

ii) a holder arranged to receive said cartridge;

iii) drive means operable to position said pipette in selected wells ofsaid cartridge;

iv) a gas pressure applicator couplable to said pipette whereby to causeliquid flow through said membrane;

v) a radiation detector operable to detect radiation from a well of saidcartridge or from said pipette; and, optionally but preferably,

vi) an electromagnetic radiation source.

Viewed from a further aspect the invention provides an assay cartridgecomprising at least two wells and a pipette positionable in at least twoof said wells, said pipette having a proximal end and a distal end, saiddistal end being closed by a liquid permeable membrane.

A pipette is a tube with an aperture at one end (the distal end) intowhich a liquid may flow on application of a reduced pressure to theother end (the proximal end). In the apparatus referred to in thepreceding paragraphs the distal end of the pipette is tipped with(closed by) a liquid permeable membrane. The proximal end of thispipette may be open or closed but if closed then clearly this must be bysome means which allows the pressure application necessary for thepipette to function as a pipette. In one embodiment described below, theproximal end of the membrane-tipped pipette is sealed with a pierceableself-sealing membrane (e.g. a rubber gasket) and pressure may be appliedthrough a hollow needle inserted through the membrane. Alternatively theproximal end may be closed by a removeable cap or stopper which isremoved to alloy pressure application, or by a frangible seal which isbroken to allow pressure application.

Viewed from a still further aspect the invention provides an assaydevice comprising a) a cartridge holder capable of receiving an assaycartridge according to the invention; b) drive means operable toposition the pipette of a said cartridge in selected wells of saidcartridge; c) a gas pressure applicator couplable to the pipette of asaid cartridge whereby to cause liquid flow therethrough; d) a radiationdetector operable to detect radiation from a well of a said cartridge orfrom the pipette thereof; and, optionally but preferably, e) anelectromagnetic radiation source.

Thus the combination of the device and the cartridge of the inventionprovides an assay apparatus according to the invention.

The assay cartridge preferably is provided to the user pre-filled withthe reagents required for the particular assay or assays to be performedusing that cartridge. Where two or more reagents are required and theseshould not be mixed before the assay is performed, these may bepre-filled into different wells in the cartridge. Generally suchreagents will be prefilled into the wells in measured quantities. Suchreagents may for example be liquids, powders, beads, coatings on thewell walls, coatings on beads, or materials impregnated into orimmobilized on the membrane of the pipette. Where the reagents areliquid or where they are susceptible to degradation on exposure to airor moisture, the cartridge may be sealed to prevent liquid loss or airor moisture access to the susceptible reagent. Such sealing isconveniently achieved by forming the cartridge with a well-containingbase and a well-covering cap, and if necessary placing a fluidimpermeable seal, e.g. an O-ring, between the well-openings in the baseand the well-covering cap, and if desired by placing a removable-seal,e.g. an adhesive sealing strip, about the external junction between capand base. In another more preferred embodiment, one or more of the wellsmay be foil sealed before use: in this embodiment the well covering capis preferably equipped with foil seal cutters for cutting thewell-covering foil seals to permit the pipette to be inserted into thesewells. Alternatively, the cap may be provided with resilient material atpositions corresponding to the tops of the wells (or just the liquidcontaining wells) such that when cap and base are urged together, aliquid-tight seal is formed at the well tops. Such material may forexample be a layer coated onto the cap or discs or gaskets attached(e.g. welded or adhered) to the cap. In one embodiment, the lowersurface of the cap is provided with resilient projections capable offunctioning as stoppers for the wells. In this way, the stoppers serveto keep cap and base together before use of the cartridge in an assayand after assay performance base and cap can be sealed for disposalsimply by urging the two together causing the stoppers again to seal thewells. This is particularly advantageous when the wells following assayperformance contain toxic or potentially infectious materials. Such capscan, if desired, be removed before use; however, in a preferredembodiment, the cap will serve to hold the pipette and possibly also toprovide attachment means for the pressure applicator. In such anembodiment the drive means may serve to move base relative to cap so asto position the pipette in the desired wells in the different stages ofthe assay.

In general, and particularly where the cartridge cap is provided withresilient stoppers for the wells in the cartridge base, the apparatusand device of the invention preferably comprise means for separating thecap from the base so that the cartridge may be loaded into the devicestill sealed. In one embodiment, such separating means comprises a wedgewhich is moved past the loaded cartridge and engages with projections,e.g. flanges, on cap and base to force the two apart. Desirably thisseparating means is automatically brought into operation followingcartridge loading, e.g. in response to the shutting of the lid to thechamber containing the loaded cartridge or on transport of the cartridgeinto the chamber for example using a conveyor which can similarly removethe cartridge from the chamber following assay performance.

For different assays, e.g. for different analytes, different assaycartridges may be provided; however, cartridges may be designed forperformance of two or more different assays. In this latter case, itwill frequently be desirable for the cartridge to contain two or moremembrane-capped pipettes, i.e. so that a different pipette can be usedfor each of the assays.

The wells in the cartridge may be provided in any desired pattern, e.g.as a two dimensional array (e.g. as in conventional multi-well plates),as a linear array, or as a circular array. The use of circular andespecially linear arrays is particularly preferred as the mechanismrequired for moving the cartridge between preset positions issimplified, i.e. the drive means may then operate to move the cartridgealong a linear path or to rotate the cartridge.

The use of a linear array of wells is especially preferred, particularlyan array comprising, in sequence: a material handling well (optionallybefore use storing a capillary-tipped pipette removably mounted on thecartridge cap or adapted to receive during use a capillary-tippedpipette mountable on the cartridge cap); a well which before use storesthe membrane-tipped pipette or a further capillary tipped pipettemounted on the cartridge cap; and one or a series of two or more (e.g.up to six) wells for assay performance and assay result reading—thesewells may contain reagents and before use such reagent containing wellsmay be foil sealed and one of these wells may be open ended oropen-sided to facilitate result reading. In such an arrangement, the capand base may desirably be separated before assay performance begins andre-engaged only when assay performance is completed. Thus result readingin this arrangement takes place while cap and base are disengaged fromeach other. In this arrangement, the cap and base are preferably latchedtogether, e.g. by a snap-lock latch. The material handling well may forexample contain dry reagent for mixing during assay performance, afilter for sample separation (e.g. to remove erythrocytes from a bloodsample), or a further pipette capable of mating engagement with acap-mounted pipette (e.g. a capillary-tipped pipette).

While the cartridge must contain at least two wells, one or morepositions in the well array of a multi-well cartridge may be open-endedor open-sided such that detection of radiation from the pipette whenlocated in such positions is facilitated. If radiation from a pipette ina well is to be detected, then at least a portion of the well wall mustbe transparent to the type of radiation to be detected.

The wells in the cartridge may remain stationary during the assay;however, as it may be desirable to use the detector to monitor theprogress of the assay, it is generally preferable that the drive meansis operable to move the cartridge between two or more pre-set positionsso that the detector can detect radiation from different cartridgewells. Alternatively but less preferably, the detector itself may bemovable between pre-set positions or movable mirrors may be provided soas to permit the light path from cartridge to detector to be varied toachieve the same effect.

Thus in a preferred embodiment the drive means will operate during theassay to lift the cartridge cap and pipette away from thewell-containing base (or more preferably to drop the base away from thecap), to move the base relative to the cap (preferably by moving thebase, e.g. linearly or by rotation) to bring the pipette into registrywith the desired well, and to move cap and base together to place thepipette into the desired well, and so on until the assay is complete.

In some assays it may be desirable to tilt the wells during liquidtransfer or to agitate liquid in a well and accordingly it is desirablethat the drive means also be operable to tilt or agitate (e.g. rock orshake) at least the well-containing portion of the cartridge.

The drive means may be manually operable, e.g. a mechanical drive or amotor driven drive activated at each stage by the operator; however itwill preferably be a motor drive activated to perform the requiredactions by an external or more preferably internal computer whichoperates the assay apparatus.

The wells in the cartridge may be of any desired shape or volume;however preferably they will be straight-sided cylindrical or lesspreferably tapered cylindrical. The cross-section of such cylindricalwells may be of any desired shape, e.g. circular, oval, polygonal (e.g.rectangular), semicircular, etc. The well bases may be flat or curved;however for wells which are to be monitored from below during or at theend of the assay, the well base will preferably be flat. In aparticularly preferred embodiment, the well base is flat and sloping,i.e. non-horizontal. The wells may be within a solid base oralternatively, and less preferably, the wells may be connected in astrip, plate, disc, daisy-wheel, etc. format. The well walls, forexample the solid well-containing base, will preferably be of plastic,especially light-transparent plastic, e.g. acrylic, vinylic, styrenic orolefinic plastic. The choice of the particular plastic will howeverdepend, as is conventional, on the nature of the reagents used. It hasbeen found particularly preferable to use plastics with good opticalproperties and low gas and/or liquid permeability. To this end,copolymers of alpha-olefins (e.g. ethylene and propylene, especiallyethylene) and cyclic olefins (e.g. norbornene) are especially preferred,e.g. the product sold under the trade name Topas® 8007 by Ticona GmbH ofFrankfurt, Germany (Topas® 8007 is an ethylene/norbornene copolymer).Desirably such copolymers have a light transmission (measured accordingto ASTM D1003 for a 2 mm wall thickness) of at least 80%, mostpreferably at least 90%; and a water vapour permeability (at 23° C. and85% RH, measured according to DIN 53122 on a 80×80×1 mm sample) of lessthan 0.2 g.mm.m⁻²d⁻¹, more preferably less than 0.05 g.mm.m⁻²d⁻¹.

Typically, the wells will have internal diameters of 3 to 20 mm,especially 5 to 15 mm, and volumes of 0.3 to 5 mL, especially 0.5 to 1.5mL.

The membrane-tipped pipette in the cartridge of the invention ispreferably cylindrical and the membrane is preferably at or morepreferably covering one end. The other, open, end is preferably shapedfor substantially gas-tight attachment to a pressure applicator. Thepipette may be of any appropriate material; however transparent plasticor glass is preferred. The membrane may be attached to the pipette inany appropriate fashion, e.g. by welding (e.g. ultrasonic or thermalwelding), adhesive, fusion of a granular membrane precursor, etc.

The membrane itself may be of any appropriate material, e.g. plastics(e.g. nylon, polysulphones, etc.), glass (e.g. glass fibre), metal, etc.However cellulosic membranes (e.g. reinforced nitrocellulose) areespecially preferred as it is relatively straightforward to immobilizeantibodies or other assay reagents on such materials.

In various embodiments of the invention, the membrane is preferablyplanar and perpendicular to the pipette axis; such membranes areparticularly effective for removal of liquid from a horizontal flat- orconcave-bottomed well.

The membrane however may alternatively and more preferably be planar butangled relative to the axis of the pipette, e.g. up to 85° offperpendicular to the axis, preferably 10 to 80° off perpendicular, morepreferably 50 to 70° off perpendicular, especially about 60° offperpendicular. Where the pipette and one or more of the wells isrectangular (e.g. square) in cross-section, it is preferred that themembrane be angled and that the base of one or more such wells likewisebe angled so as to be substantially parallel to the membrane when thepipette is in that well.

The use of a sloping membrane is especially advantageous as for a givenpipette cross-sectional area, the surface area of the membrane isincreased as it is angled progressively further from the horizontal, sogiving a larger surface area to be read or monitored during the assay.Most surprisingly, not only do sloping membranes allow essentially allof the contents of a correspondingly shaped well to be taken up throughthe membrane but also the uptake is uniform across the membrane (i.e. ifa coloured analyte becomes trapped on the membrane the membrane becomesuniformly coloured). A further advantage is that the membrane may beviewed from the side avoiding any risk of droplets of sample, reagent,etc., falling onto the apparatus optics. A still further advantage isthat the membrane may readily be illuminated without causing highincidence of the illuminant light being reflected into the lightdetector. Another advantage is that, even with a coloured sample (e.g.blood), it is possible to monitor the membrane surface through the wellside wall and thus to terminate any reaction step when the desiredchange in membrane surface has occurred as the membrane-to-well wallspacing can be less than that for a horizontal membrane in aliquid-containing well. A yet still further advantage is that theformation of bubbles between the membrane and the facing well wall isreduced relative to the case for horizontal membranes so reducing theneed to tilt or shake the cartridge base.

The use of angled membrane tipped pipettes is thought to be novel andthus viewed from a further aspect the invention provides a pipette thedistal end whereof is cylindrical and tipped by a porous membrane theouter surface whereof is angled away from the plane perpendicular to thecylindrical axis of said distal end, said pipette preferably formingpart of a diagnostic assay cartridge.

The use of a rectangular cross-section for a well is especiallypreferred as it reduces the incidence of liquid reagents being trappedat the upper end of wells by capillary effects following inversion ofthe assay cartridges during transport or storage. The corners where wellside walls meet should therefore desirably be as sharp as possible atthe upper ends of the wells, e.g. having a radius of curvature of 0.5 mmor less, e.g. 0.1 mm or less. However, to prevent liquids in the base ofthe wells “creeping” up the corners of the well, it is desirable that atthe lower end of the wells the corners should be chamfered or morerounded, e.g. having a radius of curvature of at least 0.5 mm,preferably at least 0.8 mm.

Where a well is to be used for assay reading, e.g. where the absorptionof light passing through a liquid in the well is to be measured, it isalso particularly preferred to use a rectangular cross-section well withan angled base. In this way, by appropriate masking of the section ofthe well visible to the detector, one may choose to measure lighttransmitted through the full width of the well or through a narrowerwidth at the base of the well (i.e. between a side wall and the slopingbase). Thus the light path length through the well may be increased ordecreased by moving the visible section up or down. In this way, forexample, where the optical density of the well contents is high, ashorter path length may be chosen.

Moreover, by measuring light transmission intensity at two or more pathlengths (e.g. within and above the tapered base portion of the well),the contribution of the well walls to the detected signal can bedetermined and corrected for.

Where scattered light is to be detected (e.g. where the sample beingread contains particles or agglomerates or is fluorescent orphosphorescent), it will again be desirable to use rectangularcross-section wells with the incident light being directed perpendicularto one pair of well walls and with the scattered light being detected bya detector (e.g. digital camera) directed at one of the other walls.Where the cartridge contains a linear array of wells, the reading wellfor light scattering measurements is preferably at one end of the array.

This use of angled wall wells is also novel and forms further aspects ofthe invention.

Viewed from a further aspect the invention thus provides an assayapparatus comprising:

i) an assay cartridge comprising at least one well and a pipettepositionable in at least one said well, at least one said well havingtwo parallel planar side walls joined by a base wall comprising at leastone planar face the normal to the surface whereof is coplanar to andnon-perpendicular to normals to the parallel planar surfaces of saidside walls;

ii) a holder arranged to receive said cartridge;

iii) drive means operable to position said pipette in selected wells ofsaid cartridge;

iv) a gas pressure applicator couplable to said pipette whereby to causeliquid flow through said membrane; and

v) a radiation detector operable to detect radiation from a well of saidcartridge or from said pipette.

In this aspect the base is preferably planar, angled to the horizontalas described above, and the well is preferably rectangular incross-section. The cartridge moreover preferably contains at least onecapillary-tipped pipette and/or membrane-tipped pipette, again asdescribed herein.

Viewed from a still further aspect the invention provides an assaycartridge comprising at least one well and a pipette positionable in atleast one said well, at least one said well having two parallel planarside walls joined by a base wall comprising at least one planar face thenormal to the surface whereof is coplanar to and non-perpendicular tonormals to the parallel planar surfaces of said side walls.

In addition to a membrane-tipped pipette, the cartridges of theinvention may contain one or more further pipettes, again preferablycarried by the cartridge cap, for example for measuring out an accuratevolume of reagent or sample or for mixing reagents and samples. In onepreferred embodiment the cartridge contains a capillary-tipped pipettewhich draws up a desired amount of fluid from a sample by virtue of itscapillary action. Particularly desirably this comprises a capillaryopening into a chamber of wider internal diameter such that capillaryaction causes only the capillary tip to fill. With the tip withdrawnfrom the surrounding liquid, the contents of the tip can then be ejectedinto a cartridge-well under pressure or sucked up further into thepipette beyond the capillary tip and chamber.

In another aspect of the invention, the cartridge may comprise acapillary-tipped pipette in place of the membrane-tipped pipette. Aswill be discussed further below, such a cartridge may for example beused in a clotting time assay.

The external diameter of the membrane-tipped pipette is preferably atleast 0.8 mm, e.g. 1 to 5 mm, especially 1.5 to 2.5 mm, less than theinternal diameter of the wells so as to facilitate gas flow between wellwall and pipette during liquid transfer across the pipette membrane andto ensure substantially complete uptake of liquid from the wells. Thegap also allows the well to contain liquid (e.g. 200 μL) and themembrane-tipped pipette before uptake of liquid into the pipette.

While the pipette, and the wells may have the same form ofcross-sectional shape (i.e. circular, square, etc.), it may occasionallybe preferred that the shapes differ slightly, e.g. one being circularand the other elliptical, as this reduces the risk of themembrane-tipped pipette being held by suction to the bottom of a well.This problem may similarly be addressed by making the pipette tip or thewell base slightly irregular, e.g. with indentations or projections.

In a particularly preferred embodiment, the cartridge comprises: a basecontaining a plurality, e.g. 2 to 8 or 10, of wells, at least two andpreferably at least 3 of which are free of liquid reagents and at leastone of which contains a liquid reagent; and a cap carrying themembrane-tipped pipette such that it is disposed with the membrane endin one of the empty wells and with the open end accessible on the outersurface of the cover, and having a sample application aperture throughthe cover to communicate with another of the liquid-free wells.Desirably removable seals are provided to cover the open ends of thepipette and the sample application aperture. Unless the cap carrieswell-sealing stoppers or the wells are sealed as described above, afurther removable seal will preferably be provided to surround theexternal junction of cap and base and O-ring or other seals will beprovided around at least the liquid containing wells between cap andbase. In either of these ways the interior of the cartridge is isolatedfrom air and moisture before use. The base and cap preferably haveindentations or projections for engagement with the cartridge holder anddrive means, for ensuring correct registry between cap and base duringassay performance, and if the cap carries well-sealing stoppers, forengagement with a separator such as described above which operates toseparate cap and base to allow the assay to proceed.

The base and cap are preferably such that the membrane-tipped pipettecan be placed within a “reading well” or in a well-free position atwhich radiation from the pipette is accessible to the detector. Such a“reading well” may for example have a light-transparent flat base orflat side well section through which light may pass to the detector. Inthe case where reading is at a well-free position, this may for examplebe an open-ended aperture through the base or a portion of the basewhere its side wall is removed or recessed such that light from thepipette may reach the detector without passing through the material fromwhich the base is formed.

The use of a “reading well” is preferred since the possibility ofreagents or sample dripping into the body of the assay apparatus isreduced. Where an angled membrane is to be read, the use of a separatereading well may be avoided as simply lifting the membrane out of theliquid in a well or sucking the liquid through the membrane into thepipette leaves the membrane surface exposed for reading.

In one embodiment, the base may be formed to provide a mirror surface(e.g. a plastic prism surface) under the bottom of the reading wellwhich reflects light from the bottom of the reading well, e.g. from thevertical to the horizontal. In this way, the detector need not bepositioned below the cartridge and problems of dust or liquid fallingonto the detector may be avoided. As in a Fresnel lens, a prism maysimilarly be produced as an integral combination of parallel individualprism elements. This prism structure is referred to herein as a “Fresnelprism”, and such prisms and their uses, e.g. as light path modifiers inoptical apparatus, for example assay devices, form further aspects ofthe present invention. Image distortion, due to surface distortion oftenseen in plastic mouldings with a thickness of more than few millimetres,is reduced or avoided by use of a plastic Fresnel prism rather than aconventional plastic prism having the same light incidence surface area.Thus the use of a Fresnel prism formed in the cartridge base to achievelight reflection is especially preferred in the devices of theinvention. A typical “Fresnel prism”, is a structure of transparentmaterial stepped on one side and flat on the other—light incidentnormally on the horizontal part of a step is internally reflected by theflat surface and leaves normally through the vertical part of a step. Ineffect therefore it functions as a mirror. With an angled membranehowever such a Fresnel prism will not generally be needed.

In the cartridges of the invention, the proximal or “open” end of atleast one pipette is preferably sealed with a resilient self-sealingmembrane, e.g. a rubber membrane, which may be pierced by a hollowneedle to allow gas pressure application. In this embodiment, a wastereservoir is preferably disposed in the pipette between the pipette tipand the resilient membrane. With this embodiment, liquid in thecartridge may be drawn up into the waste reservoir during or at the endof assay performance so that the used cartridge may be removed anddisposed of without waste leakage occurring.

The gas pressure applicator in the apparatus of the invention may forexample comprise a pump, and a conduit from the pump to a cartridgeattachment, and optionally at least one reservoir and a two or moreposition valve Inclusion of a reservoir, e.g. of one or more litrecapacity, and preferably at least two reservoirs, allows pressures aboveand/or below ambient to be applied to the pipette for short durationswith negligible time variation of the pressure applied due to theability to isolate the pipette from the pump and due to the relativelysmall pressure change within the reservoir during the pressureapplication period (as a result of the relatively large size of thereservoir). Between pressure applications, the pump can be used to bringthe reservoir pressure back to the desired level. Since it may bedesirable to vent the pipette to atmospheric and or to provide pressuresabove and below ambient to the pipette, it is desirable to place amulti-position valve in the conduit upstream of the pipette to allowsuch different pressure applications. The valve, which should desirablyalso include a closed position allowing no gas flow to or from thepipette, is preferably computer operated. The use of pressure reservoirsas described above however results in a relatively large spacerequirement for the apparatus and device of the invention. Since thedevice is preferably portable, it is preferred instead to use apiston-based pump (e.g. a syringe) coupled via a conduit (preferably ofminimal volume) to a cartridge attachment. Indeed it is especiallypreferred to have an array of coupled piston-pumps, each connected to aseparate cartridge attachment so that, when the cartridge is in place,operation of a pump motor causes all of the pumps to operate. In thisembodiment, the cartridge is preferably provided with blank or activemeans for engaging each of these attachments, the blank engaging meanssimply allowing the respective piston pump to vent. In certainembodiments, for example in clotting time measurements or where ananalyte is required to bind to a ligand immobilized on the pipettemembrane, it may be desirably to speed up or slow down passage of liquidunder the influence of the pressure applicator; in these circumstancesthis may for example be achieved by speeding up or slowing down thespeed of the pistons in the piston-pumps.

The pressure applicator is preferably coupled directly to the open endof the pipette; however alternatively and much less preferably it may becoupled directly to a well in the cartridge with the open end of thepipette open to ambient pressure.

In one particular embodiment, a (preferably moveable) pressureapplicator attachment is provided for each well or well-free-readingposition of the cartridge and the cartridge is provided with blank oractive means for engaging each of these attachments. In this way it maybe possible to avoid the need for careful orientation of the cartridgeduring placement in the holder—the cartridge could be placed in any oneof the pre-set permitted orientations and the lid of the apparatusclosed to bring the attachments automatically into engagement with theblank and active engagement means on the cartridge. Cartridgeidentification (as discussed further below) by the apparatus would thenallow the cartridge to be moved automatically into the correctorientation for commencement of the assay. This however is onlyespecially desirable if it is important to reduce the time required forcartridge placement or if the cartridge is designed for use in multipleassays (i.e. has multiple pipettes).

The detector in the apparatus of the invention may be any appropriateradiation detector, e.g. a radioactive emission detector or anelectromagnetic radiation detector. Alternatively the apparatus maycontain two or more detectors capable of detecting different types, ofradiation. However, for point of care use, it is preferred that thedetector be an electromagnetic radiation detector and more specificallya detector capable of detecting light in at least part of the UV to IRrange, particularly the near UV to near IR range and more especially thevisible range. (The term light is used here to mean electromagneticradiation in the UV to IR range.) For this purpose it is especiallypreferred to use a digital camera as the detector.

The use of a digital camera as the detector is especially preferredsince it can function not only as a light detector but as an imagestructure analyser. Thus, for example, irregularities in the image of amembrane on a pipette may be detected and corrected for.

Between detector and cartridge it may be desirable to place, movably orfixedly, items which serve either to select the radiation energy allowedto pass to the detector (e.g. filters, prisms, etc.) or to reduce strayradiation impact on the detector (e.g. apertures and light traps).

Stray radiation reducing items are especially important where theradiation to be detected is weak (e.g. resulting from chemoluminescenceor fluorescence) or stimulated or results from transmission orreflection of radiation measurable by the detector. In suchcircumstances, light barriers or collimators may also be providedelsewhere in the apparatus or within the cartridge.

In general, the apparatus of the invention will be provided withelectromagnetic radiation sources (e.g. sources of visible light or nearIR to near UV), disposed to cause radiation emitted, reflected ortransmitted by the desired cartridge wells or pipette to pass to thedetector. As a result it is also preferred that cartridge, cartridgeholder and detector be disposed in a light proof chamber in theapparatus and that the apparatus be provided with a closable access portfor cartridge placement, e.g. a lid.

It is especially preferred that a light source be provided which, whenthe cartridge is in place, has a well between it and the detector, e.g.so that light transmittance in the well may be determined. For thispurpose, the cartridge may be provided with an aperture into which sucha light source may be inserted on cartridge loading, preferably anaxially positioned aperture where the wells in the cartridge aredisposed about a central axis.

It will be realised that the detector may be positioned relative to welland light source so as to detect transmitted, reflected, scattered oremitted light.

Where the detector is a digital camera (or a scanning laser), it mayalso be used for assay identification. Thus a bar-code or similarmachine readable code may be placed on the assay cartridge and, readingthis the computer running the apparatus can identify the nature of theassay and hence the assay steps necessary to effect. The assay user cansimilarly apply a bar-code or machine readable code to the assaycartridge to identify the patient so that the apparatus may generate areport identifying patient and assay or may generate an entry in or forthe patient's computerized records. Code-reading and result readingsystems of this nature are discussed for example in WO 98/32004.

As mentioned above, cartridges in which the pipette is capillary-tippedrather than membrane tipped may conveniently be used for assaying forcoagulation time in blood or plasma (preferably blood). The pipetteconveniently comprises in sequence a capillary tip, a chamber and asecond capillary, which may be non-linear, e.g. sinuous, if desired.Opening the cartridge and dipping the capillary tip in a blood samplecauses it to fill up to the junction with the chamber, i.e. to take up apredetermined sample volume. The cartridge may then be closed and placedin the assay device. The second capillary or one of the wells in thecartridge coated with a clot-promoting agent (e.g. tissue factor) andthe liquid sample may be contacted with this by application ofsub-ambient or above ambient pressure respectively to the open end ofthe pipette. In the first case, the pressure causes the sample to bedrawn through the chamber into the second capillary and so into contactwith the clot-promoting agent. In the second case, the pressure appliedexpels the sample into the coated well. If desired, in this second case,the sample and clot-promoting agent may be mixed by being drawn backinto the pipette and expelled again one or more times. Thereafter thesample is drawn through the capillary tip and chamber into the secondcapillary. In both cases, the motion of the sample in the secondcapillary under applied pressure is monitored by the detector untilclotting has proceeded to the extent that motion is no longerdetectable. This may require the sample to be shuttled back and forth inthe second capillary by alternate application of below and above ambientpressures.

It will be appreciated therefore that the same capillary can be used forcollecting the sample (e.g. blood), and mixing it with one or morereagents (e.g. by pumping it into and out of a well in the cartridge).

In any event, for clot time measurements it is important for the sampletemperature to be controlled and thus it is desirable that the device,e.g. in the cartridge holder, be provided with temperature control, e.g.a thermostated hot-plate, a hot air source, etc.

In an alternative embodiment, clotting time in blood or plasma may bedetermined by depositing the sample into a well containing aneffervescent agent and monitoring the rate of rise of the bubblesgenerated using a digital camera.

Where a capillary tipped pipette is used, it may be desirable for thisto be provided separate from the cartridge, formed to be positionable ina well and couplable to the pressure applicator.

Such capillary-tipped pipettes and their use in conjunction with assaycartridges form further aspects of the invention.

Thus viewed from a further aspect the invention provides an assayapparatus comprising:

i) an assay cartridge comprising at least one, and preferably at leasttwo, wells and a pipette positionable in at least one, and preferably atleast two, of said wells, said pipette having a capillary tip;

ii) a holder arranged to receive said cartridge;

iii) drive means operable to position said pipette in selected wells ofsaid cartridge;

iv) a gas pressure applicator couplable to said pipette whereby to causeliquid flow through said membrane; and

v) a radiation detector operable to detect radiation from a well of saidcartridge or from said pipette. Viewed from a still further aspect theinvention also provides an assay cartridge comprising at least one, andpreferably at least two, wells and a pipette positionable in at leastone, and preferably at least two, of said wells, said pipette having acapillary tip.

Using the pipettes in the assay cartridges of the invention, it is thuspossible to introduce test samples into cartridge wells, to mix reagentsor reagents and sample in the wells, to transfer liquids from one wellto another, etc. By pumping liquids in and out of a pipette in one wellit is possible to improve homogeneity of mixing and by pumping liquidsback and forth across a reagent-carrying pipette membrane it is possibleto increase the extent of the reaction with the reagent. By varying therate at which a liquid is pumped across a reagent-carrying pipettemembrane it is also possible to vary the extent to which the reagentreacts. Accordingly the pipette and cartridge format gives greatversatility for assay performance.

Where the assay cartridge includes a capillary tipped pipette, e.g. forconveying blood samples, it is frequently desirable to remove excessfluid from the outer surface of the capillary. In such cases, it ispreferred that one of the wells be provided with an absorbent pipettewiper against which the capillary tip may be drawn so as to cause thewiper to absorb any fluid on the outer surface of the capillary. Thiswiper may for example take the form of an absorbent pad disposed at ornear the upper end of the well, e.g. a U shaped pad, preferably notchedat the base of the U. In such an embodiment, as the capillary iswithdrawn from the well it may be displaced sideways to engage thecapillary tip with the notch. Since such displacement may occur beforethe membrane-tipped pipette is fully withdrawn from the well in which itis disposed, it may be necessary to design the wells to prevent themembrane-tipped pipette from being driven into a well side wall. Thusthe well for the membrane tipped pipette may be made wider oralternatively its side wall may be partially removed at the upper end ofthe well.

Rather than wiping a capillary tip to remove excess sample from theoutside of the tip, an alternative is to insert the capillary tip intoan absorbent array disposed parallel with the axis of the capillary tip,e.g. absorbent fibres lying parallel to the tip or sheets of absorbentmaterial (e.g. paper) with surfaces parallel to the capillary tip axis.Since the open tip of the capillary will not contact the absorbentmaterial, the contents of the capillary are not removed while theoutside of the capillary is cleared of excess fluid. This isparticularly important with blood samples. Thus for example a 1 μLcapillary shows poor precision unless the blood sticking to the outsideof the capillary is removed. On an average a 1 μL capillary carries 0.25μL on the outside. Without removal of blood sticking to the outside a CV(coefficient of variation) of about 7-8% (volume of blood delivered) isfound. With efficient removal of blood carried on the outside the CV isreduced to 1.0-1.5%.

Where capillary wiping takes place as part of assay performance, thetime delay before wiping occurs may lead to drying of the blood on theoutside of the capillary. When this happens the blood will not all beabsorbed and may be solubilized during a subsequent dilution step. Ifthe user waits one minute from taking the blood into the capillary tostarting the instrument, the wiping off is somewhat inefficient. Waitingthree minutes means no absorption of blood at all.

It is therefore greatly preferable if capillary wiping takes placeimmediately after blood sample uptake by the capillary. This can beachieved by disposing in a capillary-receiving well of the cartridge anabsorbent array as described above, e.g. a strip of paper folded into aV-shape with the open end of the V receiving the capillary tip. Thepaper may be positioned and kept stable in the well either by using theforces of the paper pushing outwards against the well walls or ifnecessary by mounting the paper in a supporting frame. When the userintroduces the capillary holder into the cartridge, the capillary willpush the two upper arms apart and the capillary will slide down incontact with the paper on two sides opposite to each other. Thisconstruction with the paper parallel to the capillary ensures that noblood can be absorbed from the interior of the capillary and in additionthe capillary will never hit the bottom part of the folded paper. Usinga 1 μL capillary and whole blood, a CV (blood volume) of 0.75% wasachieved with this construction.

In a further preferred embodiment, the assay cartridge is provided tothe user with a capillary-tipped pipette to be used for sample takingeither loose or detachably mounted in the cartridge, e.g. in an end wellof a linear well array. In this embodiment, detachably mounted on thecapillary tip, i.e. the distance end of the pipette, is a sleeve whichclosely engages and is preferably flush with the open end of thecapillary-tip. On sample uptake by the capillary, any excess externalliquid accordingly sticks to the outside of the sleeve rather than tothe outside of the capillary proper. The sleeve is preferably provided,e.g. on its external surface, with means to engage with the inner orupper surface of a well in the cartridge (e.g. a distortable flange,etc.) so that when the loaded capillary-tipped pipette is pressed intothat well the capillary-tipped pipette can then be removed from the well(e.g. on commencement of automated assay performance) leaving the sleeveand the excess external liquid behind in the well. Experiments haveshown that, in transferring a 1 μL blood sample using such a sleeveprotected capillary, CV (blood volume) as low as those achievable withthe folded paper wiper described in the previous paragraph can beachieved.

For certain assays, it may be desirable to carry out a separation of thesample, e.g. to generate a plasma sample from an original blood sample.In such cases it may be desirable to place a filter in one of the wells.This may be removable or alternatively may form part of an integralpipette extension seated in the well. Such a pipette extension may forexample comprise a cylinder open at its upper end where it is shaped forengagement with a pipette mounted on the cartridge cap, and packed atits lower end with glass fibre. In one such embodiment, the sample maybe taken up into a capillary tipped pipette mounted on the cartridge capwhen cap and base are separated or into a capillary tipped pipettemountable in the cartridge cap. Then, with the cap and base engaged, thesample may be expelled under air pressure into the cylinder of thepipette extension; the filtrate will pass into the base of the well. Asecond cap-mounted capillary tipped pipette can then be used to draw upthe filtrate after the pipette and pipette extension have been withdrawnfrom the well. In this way, starting from a blood sample, an undilutedplasma sample may be produced.

As well as pipette extensions, capillary wipers, etc., other items maybe disposed within the wells of the cartridge. Thus for example the wellfor receiving a sampling capillary may contain a further fixed orremoveable well containing a dried reagent so that the sample and thisreagent may be mixed at the onset of assay performance.

The apparatus, device and cartridges of the invention are for use inassay methods. Such methods, using the apparatus, device or cartridgesof the invention form a further aspects of the invention.

While the invention is particularly suited for medical diagnosticassays, it can also be used for other assays, e.g. environmental,nutritional, etc., including assays of samples from manufacturingprocesses. It is particularly suitable for such uses as the cartridgesand devices can be produced sufficiently small as to be fully portable,e.g. with the maximum dimension of the device (excluding any connectorsto external equipment or power sources) being no more than 30 cm, morepreferably no more than 20 cm.

The use of membrane-tipped pipettes in assays is also novel and forms afurther aspect of the invention. Viewed from this aspect the inventionprovides an assay method wherein a liquid is transferred from acontainer into a pipette, characterised in that the end of said pipettethrough which liquid enters is sealed by a liquid permeable membrane.

Viewed from another aspect the invention also provides the use of theapparatus of the invention to assay for an analyte in a biologicalsample or for a property of a biological sample, e.g. to assay forclotting time in a blood or blood-derived sample or to assay for aprotein analyte in a body fluid or body fluid-derived sample.

Documents referred to herein are incorporated herein by reference.

Examples of apparatus and methods according to the invention will now beillustrated further with reference to the following non-limitingExamples and the accompanying drawings, in which:—

FIG. 1 is a schematic cross-section through a cartridge according to theinvention;

FIG. 2 is a schematic partial cross-section through a cartridgeaccording to the invention;

FIG. 3 is a schematic partial cross-section through a cartridgeaccording to the invention;

FIG. 4 is a schematic drawing of apparatus according to the invention;

FIG. 5 is a schematic cross-section through a cartridge according to theinvention.

FIGS. 6 and 7 show dose-response curves for the assays of Examples 1 and2;

FIG. 8 shows the results of the assay of Example 3;

FIGS. 9 to 19 are schematic views of further embodiments of cartridgesaccording to the invention in which the wells are arranged in a lineararray;

FIG. 20 is a schematic view showing how a movable magnet may be used toseparate magnetic polymer beads from a sample in a well of a cartridgeaccording to the invention;

FIG. 21 is a schematic view showing how a paper strip may be used towipe excess liquid off the outside of a capillary-tipped pipette in acartridge according to the invention;

FIG. 22 is a schematic view showing how a membrane sealed wastereservoir may form part of a pipette in a cartridge according to theinvention; and

FIG. 23 is a schematic cross-sectional side view of a capillary-tippedpipette for use in an assay cartridge according to the invention.

Referring to FIG. 1, there is shown a transparent plastic cylindricalcartridge base 1 containing cylindrical wells 2 (only two of which areshown) disposed in a circular array about cartridge axis 3. Abovecartridge base 1 is disposed cartridge cover 5. The mouths of each wellare sealed by stoppers 4 attached to the cover 5. Cover 5 also holdspipette 6, presenting a pressure applicator attachment extension 7 tothe outside of the cover and with membrane 8—tipped pipette end disposedin a well 2 of the base 1. A sample introduction port 9 is also presentin the cover 5. Port 9 and pipette 6 are kept in registry with wells 2by mating projections and recesses 10, 11, 12, 13. Similar matingprojections and/or recesses 14 (here shown as recesses) are provided inbase 1 and cover 5 to allow base and cover to engage with cartridgeholder and drive means (not shown) of the assay apparatus. Base andcover are provided with flanges 15 to engage with the separator (notshown) which pushes base and cover seals 16 apart before assayperformance begins. The nature of the assay for which the cartridge isintended is identified by a bar-code label 17 on the side of the base.The pipette and sample application port are shown sealed by removablestrip seals 16. These are removed before the cartridge is used.

In FIG. 2, the cartridge of FIG. 1 is shown in a different orientationfor assay result reading at the end of assay performance. In thisorientation, the wells 18 and 19 shown are different from the wells 2 inFIG. 1. Well 18 is a “reading well” having a plastic prism 20 placed atits base and part of the light path from membrane to detector is shownas a dotted line 21. Pipette 7 is shown as containing used reagent 22.Light source 44 is shown in place inside axial channel 45 in thecartridge base.

In FIG. 3 is shown a different embodiment of the cartridge of FIG. 2 inwhich the bottom of reading well 18 is stepped and the base belowreading well 18 is inclined whereby together to form a Fresnel prism 29.Light source 46 is arranged to illuminate the membrane. In thisembodiment, the pipette 7 is also shown with a relatively large volumechamber 47. This facilitates retention of the liquids used in the assayin the pipette.

In FIG. 4 the components of the apparatus of the invention are shownschematically. Cartridge 23 (with base 1, cover 5 and pipette 6) is heldby holder 24 and moved by drive means 25. Pipette 6 is connected viaconduits 26 to piston pumps 27 driven by motor 28. A detector, a digitalcamera 32, is arranged to detect light from the reading well ofcartridge 23 when the assay is completed and light sources 44 and 46with power supply 34 are arranged to illuminate the reading well.

Drive means 25, motor 28, camera 32 and power source 34 are operated bycomputer 35 which provides an output on monitor/printout 36 or to remotecomputer 37 (e.g. via an infra-red wireless connection). Camera 32,light sources 44 and 46, holder 24 and cartridge 23 are withinalight-tight chamber 38 provided with a cartridge loading and unloadingport 39.

FIG. 5 shows a cross-section through an alternative, capillary-tippedpipette usable in the cartridges of the invention.

Open pipette-end 39 is adapted to be attached to the pressureapplicator. The other pipette end is provided with a capillary tip 40which communicates to chamber 41 and thence via a further sinuouscapillary 42 to open-end 39. Part 43 of the base of well 2 is coatedwith a coagulation promoting agent, e.g. tissue factor. Dipping thecapillary tip 40 into blood or plasma causes a fixed volume sample to bedrawn in by capillary action. Withdrawing the pipette from the sampleand then either expelling the content into the clot-promoting agentcoated well and then sucking the sample back into the capillary orsucking the sample past the tissue factor in the capillary, hastensonset of clotting and the digital camera can be used to determine thetime at which sample flow along capillary 42 effectively ceases, i.e.the clotting time.

FIGS. 9 to 19 showed alternative arrangements for an assay cartridge inwhich the wells are arranged in a linear way.

FIG. 9 shows a detached capillary tipped pipette 50 which may be dippedinto a liquid to take up a sample. The loaded pipette may then beslotted into aperture 51 in cartridge cap 52 so disposing the capillarytip in an end well in cartridge base 53. The open upper end of pipette50 is provided with notches 54 so that if the operator engages thepipette with the cartridge cap and base by pressing on the top of thepipette this does not raise the pressure in the pipette and so expelsome or all of the sample prematurely. FIG. 10 shows the cartridge ofFIG. 9 assembled following insertion of the sampling pipette, i.e. atthe stage when the cartridge is ready to be placed in the apparatus ofthe invention.

During performance of the assay, cartridge cap and base will beseparated by disengagement of latch mechanism 84. The separatedcartridge is shown in FIG. 11. Cartridge cap 52 is shown carryingcapillary tipped pipette 50 and membrane tipped pipette 55. Membranetipped pipette 55 is rectangular in cross-section and has an angled tip56. For clarity, the membrane covering the open lower end of pipette 55is not shown. Cartridge base 53 is shown with six wells 57-62, allgenerally rectangular in cross section. To allow for capillary tipwiping, a portion of the upper section of the wall between wells 57 and58 is absent. As shown in FIG. 12, the bases 63 of wells 59 to 62 areangled so as to be parallel to the tip 56 of the membrane-tippedpipette. Wells 59 to 62 are foil-sealed at their upper ends. The foilseals are pierced during assay performance by piercers 64 initiallymounted in the cartridge cap (see FIG. 13). The individual piercers areconnected together in a strip 65 shown in FIG. 14. Each piercer, whichmay be metal but preferably is plastic, is a hollow rectangularcross-section cylinder with a blade edge 66 on the lower rim and flanges67 on the upper rim which cause the piercer to be retained by thecartridge base once it has been forced into engagement with the base (asshown in FIG. 15). The internal cross section of the piercers is shapedto act as a guide for the pipettes.

FIG. 16 shows the cartridge cap and base being separated with a sidewaysdisplacement to bring the capillary tip of pipette 50 into contact withan absorbent wiper 68 disposed at the top of well 57. As shown,membrane-tipped pipette 55 is partly displaced from well 58 into well57.

FIGS. 17 and 18 are exploded views of cartridge cap and base assemblieswith pipette extensions 69 and 70 which in use would be disposed in thewell (57) into which the sampling pipette 50 is initially introduced. Inthe case of FIG. 18, the pipette extension 70 serves to transform thesampling pipette into a membrane tipped pipette, e.g. to allow a sampleto be filtered.

FIG. 19 shows the lower ends of three wells arranged for performance ofblood clotting assays having in FIGS. 19 a and 19 b a steel ball 72movable along the base of the well and in FIG. 19 c a polymer ball 73which will float on the sample surface while it is still fluid.

After assay performance using the cartridges of FIGS. 9 to 19, anabsorbent strip is preferably inserted into aperture 71 in the cartridgecap so as to prevent seepage of any fluid remaining in wells 58 to 62.Alternatively, the aperture may be sealed with an elongate “piston”which is used to press the piercers through the foil seals of wells 58to 62.

In FIG. 20, is shown a well 75 in a cartridge according to theinvention. This well contains a liquid 76 containing magnetic polymerbeads. To separate the beads from the liquid during assay performance(e.g. as in Example 12 below), a magnet 77 is moved from a position (A)in which it is remote from the well to a position (B) in which itcontacts the well wall. A membrane-tipped pipette can then be insertedinto the well and used to withdraw the liquid leaving behind themagnetic beads.

In FIG. 21, is shown schematically a cartridge 78 according to theinvention with a linear array of wells 79-84, an end one 79 of which isarranged to receive a sampling capillary the tip 85 of which is shown.Within well 79 is disposed a V-shaped fold of absorbent paper 86 suchthat insertion of capillary tip 85 into well 79 causes the sides of thecapillary to be wiped.

In FIG. 22, is shown partially and schematically a cartridge 87according to the invention having capillary-tipped and membrane-tippedpipettes 88 and 89 in cartridge cap 90. The membrane-tipped pipette 89has towards its proximal end a liquid waste reservoir 91 and when inplace within cartridge cap 90 the reservoir is closed by a self-sealingrubber gasket 92. Where pressure is to be applied to the proximal end ofthe membrane-tipped pipette 89 this is done by piercing the gasket 92with a hollow needle 93 attached to a pressure applicator (not shown).

In FIG. 23 is shown a capillary-tipped pipette 94 which is provided aspart of an assay cartridge according to the invention. As provided tothe user, pipette 94 is loosely positioned in one well, e.g. as pipette50 in well 57 in the embodiment of FIG. 11. The distal end 95 of pipette94 is provided with a sleeve 96 which grips the pipette end and closelysurrounds and is flush with the very tip of the capillary. The upper rimof sleeve 96 is provided with a distortable flange 97 which can beforced past a matching flange in the well so as to lock the sleeve intothe well. In use, the capillary-tipped pipette is removed from thecartridge with sleeve 96 attached, dipped into a liquid sample to takeliquid into the capillary tip, and replaced in the well and pressed tolock the sleeve into the well. The cartridge may then be loaded into theassay device and in assay operation separation of cartridge cap and baseserves to disengage the sleeve from the capillary.

EXAMPLE 1 Assay for C-Reactive Protein in Serum

1 μl samples of human blood, spiked with purified C-reactive protein(CRP) to concentrations ranging from 0 to 160 mg/l are placed in a 9 mminternal diameter, round-bottomed well (in an assay cartridge equivalentto the cartridge of FIG. 1) containing 200 μL of an aqueous dilutionliquid (30 mM borate buffer, pH 8.0 containing 0.01% w/v sodium citrate,0.02% w/v NaN₃ and deoxycholate).

The membrane-tipped pipette, having an external diameter of 7.2 mm, islowered into the sample-containing well, and below ambient pressure isapplied to the open end of the pipette causing the well contents to flowthrough the membrane into the pipette. In this Example, the pipettemembrane is a nitrocellulose sheet having immobilized thereon amonoclonal anti-CRP antibody (prepared by conventional techniques).

The pipette is then removed from the well and lowered into a second wellof the same configuration containing 200 μL of an aqueous dispersion ofgold microbeads (average diameter 4.5 nm, concentration (optical densityat 540 nm) of about 3, corresponding to an antibody concentration ofabout 50 μg/mL in 50 mM borate buffer pH 8.05, containing 20 mM NaCl,0.05% w/v NaN₃ and 0.1% w/v BSA) conjugated in conventional fashion to amonoclonal anti-CRP antibody. Below ambient pressure in again applied tothe open end of the pipette causing the liquid in the well to pass intothe pipette so saturating the membrane with the gold conjugate.

The pipette is then removed from the second well and lowered into athird well, again of the same configuration, containing 200 μL of theaqueous dilution liquid (supra) Below ambient pressure is applied to theopen end of the pipette to draw the washing reagent into the pipette; inthis way, unbound gold conjugate is removed from the membrane.

The pipette is then removed from the third well and placed into afourth, 9 mm internal diameter, flat-bottomed, empty well. For thisassay, this fourth well is the reading well. The pipette membrane isilluminated (e.g. with green light from a LED) through the transparentwell-containing base of the assay cartridge and light of 540 nmreflected by the membrane is detected using a detector (e.g. a digitalcamera or a photodiode).

FIG. 6 of the accompanying, drawings shows the linear dose-response forthis assay using a green LED.

Performance of the assay requires about 40 seconds from serum additionto reflectance determination.

EXAMPLE 2 Assay for Human Serum Albumin in Urine

Human urine is depleted of human serum albumin (HSA) by ultrafiltrationand then spiked with purified HSA to concentrations between 0 and 200mg/L.

A 10 μL sample of the urine is transferred in a capillary into a 9 mminternal diameter, round-bottomed well (in an assay cartridge equivalentto the cartridge of FIG. 1) containing 200 μL of aqueous sodiumphosphate buffer, pH 5.6 containing 4.0% v/v propan-1-ol, 0.05% w/vNaN₃, 0.003% w/v Tropeolin-O and 0.5% w/v BSA. The urine is mixed withthe dilution buffer by being pumped in and out of the capillary threetimes. The capillary is removed and the membrane-tipped pipette islowered into the well. In this assay the membrane is a nitrocellulosesheet having immobilized thereon a monoclonal anti-HSA antibody. Thediluted sample is drawn into the pipette as in Example 1.

The pipette is then removed from the well and lowered into a second wellhaving the same configuration but containing 200 μL of a dispersion ofgold microbead-antibody conjugate (as in Example 1 but with an anti-HSArather than an anti-CRP antibody, 50 mM borate buffer pH 7.8, 0.05% w/vNaN₃, and 0.2% w/v BSA). The well contents are drawn into the pipette asin Example 1, and as in Example 1 the pipette is then transferred to athird (washing) and fourth (reading) well. In this assay the washingreagent is PBS, pH 7.4.

FIG. 7 of the accompanying drawings shows the dose-response curve forthis assay.

EXAMPLE 3 Assay for Glycated Hemoglobin in Blood

1 μL of whole blood is taken from a blood sample using capillary mountedon the tip of an inverted conical container, volume about 500 μL, i.e. afunnel-shaped device, to the upper end of which is attached a pressureapplicator.

The capillary is lowered into a 9 mm internal diameter, round-bottomedwell in an assay cartridge (as described for the previous Examples)containing 200 μL of an aqueous boronic acid conjugate solution.

The conjugate solution comprises 0.25 mM xylene-cyanole boronic acidconjugate (Example 18 of U.S. Pat. No. 5,631,364), 0.07% w/v TritonX-100, 9 mM zinc chloride, and 100 mM HEPES buffer, pH 8.15.

The blood sample is pumped into the well and mixed with the boronic acidconjugate solution by pumping the solution into and out of the conicalcontainer three times. The capillary is removed and the well contentsare allowed to incubate for two minutes. This permits the detergent tolyse the blood cells, the zinc to precipitate the hemoglobin and theboronic acid conjugate to bind to glycated hemoglobin.

The membrane-tipped pipette is then lowered into the well and belowambient pressure is applied causing the liquid in the well to pass intothe pipette and the hemoglobin to become trapped on the membrane. Inthis assay the membrane is a porous filter having a 1 μm pore size.

The pipette is removed from the well and placed in a second well of thesame configuration containing 200 μL of an aqueous washing reagent (50mM morpholine buffer, pH 9.5, containing 200 mM NaCl, 0.5% w/v TritonX-100, 0.1% w/v glycerol and 0.05% w/v NaN₃. Below ambient pressure isapplied to the pipette drawing the washing reagent and unbound boronicacid conjugate into the pipette.

The pipette is then removed and lowered into a 9 mm internal diameter,flat bottomed, empty reading well in the cartridge for reflectometricmeasurement of the hemoglobin trapped on the pipette membrane. Totalhemoglobin is measured using blue light at 460 nm and glycatedhemoglobin using red light at 620 nm (e.g. using red and blue LEDs). Theproportion of glycated hemoglobin relative to total hemoglobin(sometimes referred to as % Hb1Ac) is determined by the ratio of themeasured reflectancies, calibrated against samples with known % Hb1Ac.

FIG. 8 of the accompanying drawings shows the results for the assay ofthis Example for 6 blood samples analysed for % Hb1Ac 24 hours earlierusing HPLC (Variant, BioRad).

EXAMPLE 4 Liquid Collection Efficiency for Membrane-Tipped Pipettes

The efficiency of liquid collection from different well configurationswas tested for a planar nitrocellulose membrane-tipped pipette asdescribed in Example 1 in comparison with a standard conical,open-tipped pipette. In each case 200 μL of liquid was to be withdrawnfrom a flat or round bottomed 9 mm internal diameter well in a soft orhard plastic base (LDPE and polystyrene respectively). The results areset out in Table 1 below.

TABLE 1 % Liquid collected Open-tipped Membrane-tipped Well pipettepipette Soft, round 98.9 99.8 Hard, round 99.5 99.7 Hard, flat 84.0 99.5

EXAMPLE 5 Assay for Coagulation Time for Blood

The pipette of FIG. 5 is used to collect an approximately 2 μL sample ofblood. The cartridge is then reassembled and pressure is applied to thepipette to expel the blood sample into a cartridge well, the base ofwhich is coated with a coagulation promoting agent (e.g. tissue factor).Below ambient pressure is then applied to draw the sample back into thepipette, past the chamber into the sinuous capillary. The sample is thenshuttled back and forth in the sinuous capillary under the applicationof above ambient and sub-ambient pressures and, using the digitalcamera, the time between the blood sample contacting the coagulationpromoting agent and effective cessation of blood sample movement isdetermined. This may typically be about 40 seconds.

EXAMPLE 6 Assay for Coagulation Time for Whole Blood or Plasma

An assay cartridge of the type shown in FIG. 11 is used. One of wells 59to 62 contains dried tissue factor and calcium chloride or gluconate aswell as a steel ball, e.g. 2 mm diameter (see FIG. 19 a).

The apparatus into which the cartridge is to be placed is provided witha heating element to maintain the cartridge contents at about 37° C. andwith a magnet to shuttle the steel ball along the base of the well inwhich is disposed.

In well 57 is disposed a removable capillary tipped pipette capable oftaking up a preset volume of sample, e.g. 1 to 15 μL, preferably 10 μL,of whole blood, citrated venous blood, plasma or citrated plasma.

The sample is taken up by the capillary-tipped pipette which is thenplaced in the cartridge which is then placed in the assay apparatus. Thesample is then transferred into the steel ball-containing well andmixed.

The cartridge is then shuttled relative to the magnet in a horizontaldirection parallel with the tip of the ball containing well. (Either thecartridge as a whole or the magnet may be moved—however preferably thecartridge is moved with the magnet serving initially to keep the steelball static.)

A digital camera is used to monitor the position of the steel ball. Asthe mixture begins to coagulate the ball ceases to be static relative tothe magnet and this is detected by the camera so allowing the clottingtime (from contact of sample with calcium salt solution) to bedetermined.

In an alternative, less preferred, embodiment, the magnet beneath thecartridge is omitted and the ball is placed in a well with a slopingbase (e.g. as shown in FIG. 19 b). Sharp movement of the cartridge inthe direction of the lower end of the base, e.g. through mechanicalshock or by activation of an electromagnet to the side of the well,causes the ball to move up the sloping base and, before clotting occurs,the ball returns to the lower end of the base under the action ofgravity.

EXAMPLE 7 Assay for Coagulation Time for Whole Blood or Plasma

An assay cartridge as in Example 6 is used with a low density polymerball (e.g. a polystyrene ball 3-5 mm in diameter) in place of the steelball. This ball is preferably in a flat or concave-bottomed, circularcross section well (see FIG. 19 c).

A sample is taken and mixed as in Example 6 and then placed in theball-containing well where the ball will float on the sample surface.The ball is then repeatedly urged under the sample surface and allowedto float back up to the surface. As the sample coagulates the ball willreturn to the surface more slowly and then not at all.

The ball may be urged under the surface by pressure from the tip of thepipette or alternatively a magnetically movable ball may be used and amagnetic field may be switched on and off to draw the ball down andrelease it respectively. Such magnetically responsive balls may beprepared for example by depositing superparamagnetic crystals in thepolymer ball (e.g. as in the magnetic beads sold by Dynal Biotech, Oslo,Norway).

EXAMPLE 8 Assay of Clotting Time for Plasma

An assay cartridge similar to that shown in FIG. 11 is used. As inExample 6, one of wells 59 to 62 contains a citrate buffer, anothercontains fibrinogen and coagulation factor V and a third a calcium saltsolution. Well 57 contains a capillary-tipped pipette and well 58contains a filter extension as shown in FIG. 18.

A sample is taken up in the capillary tipped pipette which is thenplaced in well 57 and the cartridge is placed in the assay apparatus andthere warmed to 37° C. The sample is then transferred to thebuffer-containing well and mixed. The whole or a preset proportion ofthe mixture is then transferred into the filter pipette extension andcell-free diluted plasma is pumped into the base of the well. Apredetermined volume of cell-free plasma is then transferred into thefibrinogen containing well using a further capillary tipped pipette andthis further pipette is also used to transfer a predetermined volume ofthe calcium salt solution to the fibrinogen/plasma containing well toinitiate the clotting reaction. The well is illuminated and a digitalcamera is used to record the turbidity of the mixture in the well. Thetime from calcium addition to increase of turbidity to a pre-definedvalue is taken as the clotting time.

EXAMPLE 9 Assay for Clotting in Whole Blood or Plasma

An assay cartridge similar to that shown in FIG. 11 and described inExample 8 is used. As in Example 8, one of wells 59 to 62 containscitrate buffer and another a calcium salt solution, however theball-containing well is omitted and in place of coagulation factor V andfibrinogen the “reagent” well contains a dried thrombin-specificchromogenic substance (e.g. Nycotest Chrom (described in Janson et al.Thrombostasis and Haemostasis 62: 530 (poster 1677) (1989) and Jonker etal. Research in Clinic and Laboratory 20: 45-57 (1990)) or one of thechromogenic substances discussed in DE-A-3113350, DE-A-3413311,DE-A-3311287, U.S. Pat. No. 4,458,015 or U.S. Pat. No. 4,784,944).

The sample is taken and mixed analogously to the procedure in Example 7.The coagulation process results in thrombin formation and thus therelease of a dye from the chromogenic substance (e.g. yellowpara-nitroaniline from Nycotest Chrom).

The change in colour of the sample is followed using the digital cameraand the clotting time is taken as the time from calcium addition to apredetermined colour change.

EXAMPLE 10 Assay for C-Reactive Protein (CRP) in Whole Blood UsingEnzyme Conjugate (ELISA)

Using the capillary-tipped pipette of the cartridge, 1 μL whole blood isadded to a well (e.g. well 59) of a cartridge similar to that shown inFIG. 11 and containing 200 μL of a dilution and lysing liquid (30 mMborate buffer pH 8.0 containing 0.01% w/v sodium citrate, 0.02% w/v NaN₃and dexoycholate). The wells of the cartridge have a rectangular crosssection with inner dimensions 6.0 by 6.5 mm. The planar bottom of thewell is angled 30 degrees to the length axis of the well.

The rectangular membrane-tipped pipette (which has outer dimensions 3.7by 4.2 mm and is equipped with an anti CRP antibody-coatednitrocellulose membrane mounted 30 degrees to the length axis of themembrane tube) is lowered into the well and the lysed blood cellsolution is absorbed through the membrane by applying below ambientpressure to the interior of the membrane-tipped pipette. When all liquidis absorbed, an above ambient pressure is applied to force the liquid asecond time through the membrane and back into the well. Passing the CRPsolution twice through the membrane increases further the captureefficiency of CRP.

Subsequently the membrane-tipped pipette is moved to a similar well(e.g. well 60) in the cartridge which contains a solution of alkalinephosphatase (ALP) conjugated to an anti CRP antibody (approximately 40μg/ma ALP and 40 μg/ml antibody in 50 mM borate buffer pH 8.0 containing0.02% w/v NaN₃ and 0.5% w/v BSA. The conjugate solution is absorbedthrough the membrane and pumped back into the well by applying asequence of below and above ambient pressure inside the membrane-tippedpipette as described above for the antigen capture.

In the next step, the membrane-tipped pipette is moved to a further well(e.g. well 61) in the cartridge which contains 200 μL of washingsolution (50 mM borate buffer pH 8.0 containing 0.01% w/v NaN₃, 0.5 w/vBSA and deoxycholate) which is absorbed and subsequently pumped backinto the well. This washing step is repeated twice by moving themembrane-tipped pipette to two additional wells (not shown in FIG. 11but equivalent to well 61) which also contain the washing solution. Thetotal of three washing cycles ensures an efficient removal of unboundconjugate.

Finally the membrane-tipped pipette is moved into a still further well(e.g. well 62) in the cartridge which contains 300 μL of a solution ofthe alkaline phosphatase substrate para nitrophenyl phosphate (1.0 mg/mlpNPP in 1.0 M diethanolamine buffer pH 9.6 containing 0.5 mM MgCl₂ and0.025% w/v NaN₃). The yellow enzyme product para-nitrophenol isdeveloped by pumping the substrate solution in and out of themembrane-tipped pipette over a period of two minutes. The incubation isterminated by pumping all liquid back into the well and raising themembrane-tipped pipette out of the substrate solution. Using 300 μL ofsubstrate solution the filling height is about 3 mm above the top of theangled part of the well, thus allowing the colour to be measured throughparallel walls of the well.

With the membrane-tipped pipette raised, the absorbance is measuredusing a blue LED as a light source and a digital camera for measurementof transmitted light.

EXAMPLE 11 Assay for C-Reactive Protein (CRP) in Whole Blood Using LightScatter Measurement of Aggregated Latex Beads

Using the capillary-tipped pipette of the cartridge, 2 μL whole blood isadded to a well (e.g. well 62) of a cartridge similar to that shown inFIG. 11 and containing 120 nm Latex beads (0.2% w/v) suspended in 300 μL50 mM borate buffer pH 8.0 containing 0.01 w/v sodium citrate, 0.02% w/vNaN₃ and deoxycholate. The beads are coated by simple adsorption withanti CRP antibodies. The well has a rectangular cross section and is atthe end of the cartridge to facilitate the measurement of light scatter.Light is directed onto one side wall of the well. After an initial phaseof cell lysis which takes about 10 seconds, the increase of lightscatter is measured at an angle of 90 degrees to the incident light. Theincrease of light scatter due to the CRP-mediated aggregation of theLatex beads is measured by the digital camera at a wavelength of 425 nm.

EXAMPLE 12 Assay for Albumin in Urine Using Magnetic Beads, ColouredLatex Beads and Relectometry

Using the capillary-tipped pipette of the cartridge, 2 μL urine is addedto a well (e.g. well 62) of a cartridge similar to that shown in FIG. 11and containing 1000 nm magnetic polymer beads (0.2% w/v) and 1000 nmblue Latex beads (0.2% w/v) in 200 μL 30 mN sodium phosphate buffer pH5.7 containing 0.5% w/v BSA and 0.05% w/v NaN₃. The magnetic beads (e.g.of the type available from Dynal Biotech, Oslo, Norway) are coated withan antibody reacting with an epitope on the albumin molecule differentfrom the epitope recognized by the antibody coated onto the Latex beads.

After incubation for 60 sec, a Neodymium magnet (10×7×2 mm) is movedfrom its resting position (20 mm from the nearest wall of the well)'towards the well to bring the magnet in direct contact with the sidewall of the well. The magnet makes contact with the wall opposite to theangled one and covers the liquid filled part of the well (200 μL). Thewell and the positioning of the magnet are shown schematically in FIG.20. In the resting position the magnetic field working on the magneticbeads is too weak to move the beads. When in contact with the well, thedistance from the magnet to the nearest and remotest inner wall of thewell is 0.8 mm and 6.3 mm respectively. At this distance the beads arequantitatively collected on the wall after 30 sec. In the presence ofanalyte, blue Latex is linked to magnetic particles and the reactedfraction of the Latex beads will be collected on the wall whileunreacted Latex particles will remain suspended.

With the magnet in contact position, the capillary-tipped pipette isused to suck up the liquid containing the unreacted Latex particles. Themagnet is then moved away from the well to its resting position.

The capillary-tipped pipette tube is then moved into an empty well (e.g.well 61) and the liquid is delivered to this well by applying aboveambient pressure to the interior of the pipette.

The capillary-tipped pipette is then moved to a further well (e.g. well60) which contains 500 μL of washing solution (PBS, pH 7.4) and 200 μLis taken up. The capillary-tipped pipette is then moved back to the wellcontaining the magnetic beads and the beads are suspended by pumping thewashing solution in and out of the well five times. The magnet is movedinto the contact position and the magnetic beads are allowed to becollected on the wall of the well. After 30 sec the washing solution istaken back into the capillary-tipped pipette. The magnet is subsequentlymoved back to its resting position.

The capillary-tipped pipette is in the next step moved to the wellcontaining the first supernatant (well 61) and pumped into this well.

The capillary-tipped pipette is subsequently moved to the wellcontaining the washing solution (well 60) and 200 μL are taken up.

The capillary-tipped pipette is moved to the well containing themagnetic beads (well 62) and the beads are resuspended by pumping thewashing solution in and out 5 times.

A membrane-tipped pipette equipped with a 0.45 μm microporous membraneis moved to the well containing the suspended magnetic beads (well 62)and the beads are collected onto the membrane by suction.

The membrane-tipped pipette is raised out of well 62 an blue Latexparticles and the yellow-brown magnetic bead are quantified byreflectometry using a red LED for the blue Latex beads and a blue LEDfor the magnetic beads. The amount of absorbed red light/amount ofabsorbed blue light is a measure of the fraction of blue Latex in themixture and hence a measure of the amount of albumin present in thesample.

The same cartridge may also be used for determination creatinine contentof urine and hence the albumin:creatinine ratio in the urine sample.Albumin in urine provides an indicator of kidney function and thealbumin:creatinine ratio may be used to correct for diuresis.Albumin:creatinine measurement is described for example in U.S. Pat. No.5,385,847.

In this embodiment a fraction of the urine sample is mixed with adilution reagent and an enzyme or enzyme mixture which reacts withcreatinine to generate a coloured analyte which is detected using adigital camera by measurement of light transmission through a wellcontaining urine, enzymes and dilution reagent.

1-48. (canceled)
 49. An assay cartridge comprising at least two wellsand a membrane tipped pipette positionable in at least two of saidwells, said pipette having a proximal end and a distal end, said distalend being closed by a liquid permeable membrane.
 50. An assay cartridgeas claimed in claim 49 wherein the distal end of said membrane-tippedpipette is closed by a sloping liquid permeable membrane.
 51. An assaycartridge as claimed in claim 50 wherein said membrane lies in a planeat an angle of 20 to 40° to the axis of said membrane-tipped pipette.52. An assay cartridge as claimed in claim 49 wherein saidmembrane-tipped pipette has a membrane-tipped end of rectangular crosssection.
 53. An assay cartridge as claimed in claim 49 wherein saidcartridge comprises detachable base and cap members, said wells beingdisposed in said base member and said cap member being arranged to carrysaid membrane-tipped pipette.
 54. An assay cartridge as claimed in claim53 wherein said cap member comprises means to receive a capillary-tippedpipette.
 55. An assay cartridge as claimed in claim 53 wherein at leastone of said wells is sealed at its upper end by a frangible seal andwherein said cap member is provided with a cutter arranged to piercesaid seal.
 56. An assay cartridge as claimed in claim 53 wherein saidbase member comprises an absorbent wiper arranged to wipe the outside ofa capillary-tipped pipette inserted therein.
 57. An assay cartridge asclaimed in claim 49 wherein the proximal end of said membrane-tippedpipette is closed by a piercable self-sealing membrane.
 58. An assaycartridge as claimed in claim 49 wherein the wells in said cartridge arearranged in a linear array.
 59. An assay cartridge as claimed in claim49 wherein the base wall of at least one of the wells is planar andnon-perpendicular to the contiguous side walls of the well.
 60. An assaycartridge as claimed in claim 49 wherein at least one of the wells hastwo parallel planar side walls, joined by a base wall comprising atleast one planar face a normal to the surface whereof is coplanar to andnon-perpendicular to normals to the parallel planar surfaces of saidside walls.
 61. An assay cartridge as claimed in claim 49 wherein atleast one of said wells contains an assay reagent.
 62. An assay devicecomprising a) a cartridge holder capable of receiving an assay cartridgeaccording to claim 49; b) drive means operable to position themembrane-tipped pipette of a said cartridge in selected wells of saidcartridge; c) a gas pressure applicator couplable to the membrane-tippedpipette of a said cartridge whereby to cause liquid flow therethrough;and d) a radiation detector operable to detect radiation from a well ofa said cartridge or from the membrane-tipped pipette thereof.
 63. Anassay device as claimed in claim 62 wherein said radiation detectorcomprises a digital camera.
 64. An assay device as claimed in claim 60further comprising a light source arranged to illuminate said cartridge.65. An assay device as claimed in claim 62 further comprising a magnet.66. An assay device as claimed in claim 62 further comprising a heaterarranged to heat said cartridge.
 67. An assay device as claimed in claim62 further comprising a controller arranged to control assayperformance.
 68. An assay device as claimed in claim 62 wherein said gaspressure applicator comprises a piston disposed within a cylindricalhousing and a drive motor arranged to drive said piston.
 69. An assayapparatus comprising an assay device comprising: a) a cartridge holdercapable of receiving an assay cartridge according to claim 49; b) drivemeans operable to position the membrane-tipped pipette of a saidcartridge in selected wells of said cartridge; c) a gas pressureapplicator couplable to the membrane-tipped pipette of a said cartridgewhereby to cause liquid flow therethrough; d) a radiation detectoroperable to detect radiation from a well of a said cartridge or from themembrane-tipped pipette thereof; and a removable assay cartridgecomprising at least two wells and a membranetipped pipette positionablein at least two of said wells, said pipette having a proximal end and adistal end, said distal end being closed by a liquid permeable membrane.70. An assay for an analyte in a biological sample or for a property ofa biological sample which comprises assaying a biological sample in theapparatus of claim
 69. 71. An assay as claimed in claim 70 to assay forclotting time in a blood or blood-derived sample.
 72. An assay asclaimed in claim 70 to assay for a protein analyte in a body fluid orbody fluid-derived sample.